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Lipids and lipid-anchored proteins and their roles in membranes

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Hugo Spink

on 23 March 2015

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Transcript of Lipids and lipid-anchored proteins and their roles in membranes

LIPIDS in the membrane
In animal membranes, the ratio of lipid to protein, by mass, is about 50:50. the majority of the lipids are PHOSPHOLIPIDS, but also GLYCOLIPIDS and CHOLESTEROL.
Phospholipids are amphiphilic wand the most abundant lipid in animal membranes. They are formed of a hydrophilic head group and a hydrophilic tail group that varies between phospholipids and the subgroups. The main phospholipids found in membranes are phosphoglycerides, but sphingolipids are also a major part.
Lipid anchored Proteins
Protein Attachments
Proteins form covalent linkages to
three main
types of lipid.
groups such as farnesyl and geranylgeranyl residues. The protein that undergoes modification here is describes as a
protein (OpenLearn, 2015)
Lipids and Lipid- Anchored Proteins and their Roles in Membranes.
Fluidity and bilayer
Lipid based on a steroid nucleus.
It is a steroid built from 4 linked hydrocarbon rings. The steroid is linked to a hydrocarbon tail at one end, and a hydroxyl group at the other.
In membranes cholesterol is parallel to the fatty acid chains of phospholipids. The hydroxyl group of cholesterol interacts with the nearby phospholipid heads.
No cholesterol is found in prokaryotes. It makes up nearly 25% of membrane lipids in nerve cells but is absent from some intracellular membranes.
et al
. 2002
Sugar containing lipids. Glycolipids in animal cells are derived from sphingosine. The amino group of sphingosine is acylated by a fatty acid unit. One or more sugars are attached to the primary hydroxyl group.
Cerebroside is the most simple glycolipid, with only one sugar unit (glucose or galactose) at its primary hydroxyl group.
Gangliosides are more complex glycolipids and contain a branched chain of sugar units up to seven residues long.
Glycolipids are oriented asymmetrically. The sugar residues are always on the extracellular side of the membrane.
There are around 5 million lipid molecules in a 1μm x 1μm space, which equates to about a billion molecules of lipid in a plasma membrane of a typical mammalian cell. All membrane molecules are amphiphillic, having a hydrophobic nonpolar end and a hydrophilic polar end. (Alberts, 2008)
Bilayer composition
Major lipid type defining the lipid bilayer is the glycerol based phospholipid. The fatty acid tails of membrane phospholipids are usually 14-24 carbons in length, one of which usually is unsaturated (cis-double bonds) whilst the other is unsaturated. Cis-double bonds create kinks in the tail, thus their presence in the membrane defines how well phospholipid molecules cluster together

The main phospholipids in cell membranes are the phosphoglycerides. Examples of these include phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (Alberts, 2008)
In lipid anchored proteins, the protein is linked to a certain fatty acid like myristate and palmitate. These anchor the protein to the cell membrane. (OpenLearn, 2015)

Common sites of protein attachment are N-terminal groups like glycine or cysteine side chains. (OpenLearn, 2015)
The actual function of the anchored proteins remains an area of debate. It's speculated they may act in signal transduction, targetting proteins to the cell surface. (OpenLearn, 2015)
Proteins can also attach to
fatty acyl
groups like myristoyl and palmitoyl residues. The protein that undergoes modication here are described as being
fatty acylated
(OpenLearn, 2015)
GPI anchors
are glycolipids that are linked to the
Carboxy terminus
of a protein during post-translational modification (OpenLearn, 2015)
Prenylation and fatty acylation localise proteins to the cytosolic side of the PM. GPI anchors however are localised extracellularly. (OpenLearn, 2015)
Prenylation of proteins most commonly involves a
linkage to a cysteine residue near the Carboxy terminus of the protein. The cysteine is always the last on the carboxy terminus and is the fourth last residue from the end. The next two residues are aliphatic (non-polar). (OpenLearn, 2015)

Fatty Acylation
Fatty acylated proteins arise when the fatty acid groups linked to the protein are either
myristic acid
(C14) or
palmitic acid
(C16). Both are saturated lipids, and an
linkage connects the myristic acid to the the amino group of the protein, palmitic acid is connected via a specific cysteine residue using a
bond. (OpenLearn, 2015)
Myristoylation usually occurs during the translation of a protein and tends to last the the entire lifespan of the protein (OpenLearn, 2015)
Palmitoylation of a protein however happens in the cytoplasm after and is a reversible process. This reversibility is useful for signal transduction. (OpenLearn, 2015)

GPI Anchor
two fatty acyl chains, linked via a glycosidic bond to a tetrasaccharide, the other end of which is linked to a phosphoethanolamine by a phosphoester bond. The linkage between the GPI unit and the protein occurs by a amine link between the amino group of the phosphoethanolamine and the C-terminus of the protein. The fatty acyls and the sugars that make up the tetrasaccharide vary with the protein that is being modified using this process. GPI anchors are added to new proteins. (OpenLearn, 2015)

Prenylation- Motifs

The motif is thus CaaX where X is any amino acid and a denotes aliphatic residues. The type of isoprenoid group that binds the protein is determined by the variable amino acid; (OpenLearn, 2015)

If X is Ala, Met or Ser, a farnesyl group is added at the Cys, whereas, if X is Leu, a geranylgeranyl group is added. When the isoprene is added, the aaX motif is then is proteolytically removed and the C-terminus is esterified with a CH3 group. (OpenLearn, 2015)
Bilayer Formation
Amphiphilic nature of the phospholipid molecules causes them to spontaneously arrange into bilayers in aqueous environments. Hydrophobicity of molecules forces water into cage like structures around the hydrophobic molecules. Lipid molecules organize themselves such that the hydrophobic regions are buried away from the water, and the hydrophilic regions are exposed to the water. They can either form sphere shaped micelles, or form double layered bilayers. (Alberts, 2008)
Bilayer 2-D structure and fluidity
Studies on synthetic lipid bilayers have shown that phospholipid molecules that make up the lipid bilayer rarely leave the monolayer and cross from one side to the other. This is known as
, and is rare but can happen, with occurrences of around once a month or longer. Cholesterol however, can flip-flop very easily. Lipid molecules themselves flip-flop rapidly,at a rate of approximately 100 million exchanges a second! Computer studies on the lipid bilayer show that it is a very fluid two-dimensional construct with very fast lateral diffusion of it’s components. (Alberts, 2008)

A problem with the formation of bilayers is that newly synthesized molecules that will form the bilayer are done so in the cytosolic layer. E.g, phospholipids synthesized in the cytosolic layer of the ER must be able to diffuse to the non-cytosolic side otherwise a bilayer would never be able to form.
Phospholipid translocators
are proteins that catalyze accelerated flip-flopping of phospholipids between the two monolayers, solving this problem. (Alberts, 2008)

Fluidity and Composition
The fluidity of a bilayer also depends on the components that make it up.
is also a huge factor here, with studies on synthetic bilayers showing interconversion between liquid and crystalline states at certain freezing points. (Alberts, 2008)
The temperature at which this happens is known as the
phase transition
, and differs with membrane composition; the temperature is lower in the presence of more double bonds and shorter chains (Alberts, 2008)
controls the property of the lipid bilayers; it's known to improve the permeability of the bilayer. It's 4 rigid steroid rings immobilize regions of the bilayer and makes the lipids of the membrane pack closer together (Alberts, 2008)
The Inositol lipids
The inositol phospholipids comprise a small number of lipids in the membrane but they are essential components of various cell signalling and membrane trafficking pathways (Alberts, 2008)

Fatty Acids

These comprise a hydrocarbon chain that is attached to a carboxylic acid. Fatty acids usually have double bonds in the cis configuration, and most natural fatty acids have an equal number of double bonds. Each cis double bond causes the formation of a kink in the fatty acid chain that limits rotation around the C-C double bond.
Caveolae are a type of lipid that is are associated with endocytosis.
Caveolae are invaginations of the cell membrane 50-100 nanometers in width, rich in proteins and lipids (Anderson 1998).
Caveolae formation
The caveolin proteins form oligomers in the innermost section of the plasma membrane, containing 14-16 monomers, and link with both cholesterol and sphingolipids in the cell membrane, which stabilises the oligomers (Doherty and McMahon 2009).
Have a sphingoid base, usually made of sphingosine but also sphinganine or phytosphingosine. These are long integrated fatty chains and then, depending on the lipid being made, a specific fatty acid tail is attached by an amide bond to carbon 2. Sphingolipids are very complex and this arises due to the fact that there are at least 5 sphingoid bases that can be used, and approximately 20 fatty acid tails that can be attached. To make glycosphinoglipids, 500 different carbohydrates can be attached.
sphingolipids are now known to act as both first and second messengers in a variety of signalling pathways, and second, they have vital roles in membrane microdomains, the so-called 'lipid rafts'.
For extra reading see Futerman and Hannun (2004) The complex life of simple sphingolipids. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299119/
Lipid Rafts
Lipid rafts are cholesterol and sphingo-lipid enriched membrane microdomains that concentrate and segregate proteins within the plane of the bilayer.
Within any part of a membrane, lipids and proteins will not be distributed and lipid phase segregations are rare and instead dynamic regions form, where protein-protein interactions hold these regions in shape. The formation of the rafts help to concentrate and organise proteins for transport or signalling. One example of lipid rafts are caveolae.
Lipid Signalling
Many lipids function in signalling pathways, allowing eukaryotic organisms to coordinate cellular activity and respond rapidly to influences in their environment. In particular, lipids play key roles in signal transduction, allowing chemical messages to be relayed across the plasma membranes of cells and organelles in both plants and animals. Lipids can function directly as messengers, or as mediators. Often, enzymatic cleavage of lipids serves to control signal transduction. For example, phospholipids are targeted by a family of enzymes called phospholipases (
), with different enzymes in the family having different catalytic activities, or cleavage sites.

The activity of one of these enzymes in particular - phospholipase C (PLC), has been extensively studied, functions in a wide range of biological processes, and forms a good example of the important role of lipids in signalling pathways (Eyster 2007).

Phospholipase C
PLC is a membrane bound protein that catalyses the cleavage of the phosphate head from phospholipids to produce a polar phosphate group, which is released into the cytoplasm, and a molecule of diacylglycerol (
), which remains bound to the membrane. These molecules can then function as secondary messengers in two different locations in the cell. The activity of
itself is regulated, with activation of its various isoforms stimulating lipid-mediated pathways in the body. By investigating the
, as well as an example of its
cellular activity
, we can get a good sense of the complex role of lipids in cell signalling (Eyster 2007).
PI (4,5)-biphosphate (PIP2)
is a phospholipid in the cellular membrane, and a target of
in the ‘
IP3/DAG pathway
’ - named after the two molecules that are produced in the
catalysed reaction. The pathway serves to stimulate the release of calcium from the ER into the cytosol, and activate a protein kinase called
. Cleavage of
releases the phosphate head into the cytosol ( PI (1,4,5)-triphosphate (
) ), leaving
in the membrane. IP3 diffuses through the cytosol to the ER, stimulating the release of stored Ca2+, which can alter the activities of calcium-sensitive intracellular proteins.
is one of these proteins, and binding of calcium causes it to translocate to the inner-face of the cell membrane, where
, the second messenger, can activate it. Having been activated,
is now free to phosphorylate its target proteins. These can vary depending on cell type, which allows an organism to coordinate cellular action: via
pathways, different responses can be elicited from specific cells. The
pathway functions in a wide range of biological processes, from smooth muscle contraction, to fertilization (Alberts et al., 2015).

Activation of PLC
Many extracellular ligands can stimulate the activation of PLC, including growth factors and prostaglandins, but stimulating ligands can vary from cell to cell. The ability to activate PLC in response to different ligands in different cells allows an organism to elicit specific responses to stimuli with a high degree of control.

In essence though, method of activation remains the same. The stimulating ligand binds to the extracellular extension of a G-coupled protein receptor embedded within the cell membrane. This receptor facilitates the activation of a bound Gaq protein on the intracellular face of the membrane, which in turn activates PLC. PLC is now free to stimulate PLC-dependent pathways within the cell. In this way, extracellular messages are transduced across the membrane, enabling intracellular responses to be coordinated across the body (Alberts et al., 2015).
Target Example
et al.
Adapaproject.org, (2015). HomePage | The Adapa Project. [online] Available at: http://adapaproject.org/admin/tiki-index.php [Accessed 23 Mar. 2015].

Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., Walter, P. (2015). Molecular Biology of the Cell (6rd edn). (Garland Science, New York, 2015)

Alberts, B. (2008). Molecular biology of the cell, 5th edition. New York: Garland Science.

Anderson, R. G. W. (1998). The Caveolae Membrane System. Annual Review of Biochemistry 67:199-225.

Berg, J. M., Tymoczko, J. L. and Stryer, L. (2012). Biochemistry. 7th ed. New York: W. H. Freeman and Company.

Eyster, K. M. (2007). The membrane and lipids as integral participants in signal transduction: lipid signal transduction for the non-lipid biochemist. Advances in physiology education, 31(1), 5-16.

Futerman, A. H. and Hannun, Y. A. (2004) The Complex life of simple Sphingolipids. EMBO. 5:777-782.

OpenLearn, (2015). Proteins. [online] Available at: http://www.open.edu/openlearn/science-maths-technology/science/biology/proteins/content-section-2.4.2 [Accessed 23 Mar. 2015].

Glycerol based phospholipids that are the main component of biological membranes. The structure contains an alcohol (glycerol) and two fatty acids joined by ester bonds. Consists as a small polar head with two long hydrophobic side chains. This results in a stable structure. Some examples of phosphoglycerides include Plasmalogens and Phosphatidates.
They have a glycerol base with two fatty acid tails and a phosphorylated alcohol attached. Phosphatidate is the most basic phosphoglycerol and the basis for the others. By addition of alcohols to phosphate generates many more phosphoglycerides such as these (Berg et al. 2002).
In this prezi we cover the
of lipids and lipid-anchored proteins in plasma membranes, and introduce their important functional roles in
cell signalling

Berg et al 2002
(Extra reading: Sorrentino V, Rizzuto R. Molecular genetics of Ca2+ stores and intracellular Ca2+ signalling.)
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